Truncated 24kDa basic fibroblast growth factor

ABSTRACT

The invention relates to fragments of an amino acid sequence of mature, full length  24  kDa fibroblast growth factor- 2  or an analog thereof. The fragments have an activity that inhibits the migration of cultured cells as well as inhibiting angiogenesis, tumor growth, or any other processes that involve the migration of cells in vivo. This fragment does not stimulate the proliferation of cells which is in contrast to activity shown by the mature, full-length  24  kDa fibroblast growth factor- 2 . The present invention also relates to a DNA molecule encoding the fragment, an expression vector and a transformed host containing the DNA molecule, and a method of producing the protein by culturing the transformed host. Moreover, the present invention relates to a therapeutic composition the  24  kDa fibroblast growth factor fragment and a pharmaceutically acceptable carrier.

This application is a continuation-in-part of application Ser. No.10/408,415 filed Apr. 7, 2003, entitled “Truncated 24 kDa BasicFibroblast Growth Factor”, now U.S. Pat. No. 7,432,243, which claimspriority to U.S. provisional application No. 60/370,212, filed Apr. 8,2002, entitled “Truncated 24 kDa Basic Fibroblast Growth Factor (24 kDaFGF-2) Which Inhibits Cell Migration” both of which are herebyincorporated by reference in their entirety.

This invention was funded in part by grants and contracts from theNational Heart, Lung, and Blood Institute, National Institutes ofHealth, which provides to the United States government certain rights inthis invention.

A portion of the work described herein was supported by Grant No.CA-81209 from the National Institute of Health (NIH). The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced, manyreferenced by numbers in parenthesis. Full citations for thesepublications are provided at the end of the Detailed Description of theInvention. The disclosure of these publications are hereby incorporatedby reference, in their entirety, in this application.

The invention is in the field of biochemistry and medicine relates tothe 24,000 Dalton form of fibroblast growth factor-2 (24 kDa FGF-2).Specifically, this invention relates to any and all portions of the 24kDa FGF-2 that inhibit the migration of eukaryotic cells but lack thegrowth promoting activity associated with full length 24 kDa FGF-2.

Polypeptide growth factors stimulate the growth and migration of avariety of cells (1). One of these polypeptide growth factors thatpromotes endothelial cell growth, migration, and invasion is basicfibroblast growth factor (FGF-2) (2-5). FGF-2 is part of a large familyof fibroblast growth factors consisting of at least 9 separate geneproducts, which share a common domain. The single copy gene for FGF-2encodes for multiple forms of the protein of 24, 22.5, 22, and 18 kDawith the three higher molecular weight FGF-2s (“hmwFGF-2”) produced byinitiation of translation at CUG initiation sites upstream from the AUGcodon (FIG. 1) (6;7). The 24 kDa FGF-2 form is comprised of the 18 kDaFGF-2 with an additional 55 amino acids on the amino terminal end. Thestructure of the mRNA indicates that its synthesis is translationallycontrolled. The cellular localization and apparent functions of 18 kDaand hmwFGF-2 differ. The 18 kDa FGF-2 is mostly cytoplasmic and isexported to the cell surface where it is localized to the basementmembrane or extracellular matrix in association with matrix heparins andheparans (8;9). In contrast, undetectable or extremely low levels of hmwFGF-2 are present in the media of the cultured cells studied to date.Instead, the majority of the cellular hmwFGF-2 is directly translocatedinto the nucleus (10;11). The residues associated with nucleartranslocation are RG repeats found at several sites within the aminoterminal region of hmwFGF-2 (12). Thus, 18 kDa FGF-2 has been consideredto be an external regulator of endothelial cell behaviour while thehmwFGF-2 is thought to generate intranuclear autocrine signals.

We demonstrated that exogenously applied recombinant 24 kDa FGF-2 couldregulate cell behavior in two ways, stimulation of cell proliferationand inhibition of migration (13). The increase in proliferation wascomparable to that promoted by 18 kDa FGF-2 indicating that thestimulation was independent of the additional amino terminal peptide. Onthe other hand, the effect on migration was opposite to that of 18 kDaFGF-2. While 18 kDa FGF-2 promoted cell motility, 24 kDa FGF-2 inhibitedmigration of endothelial cells by 50% and mammary carcinoma MCF-7 cellsby greater than 70%, even in the presence of unrelated mitogens thatpromote cell migration such as vascular endothelial growth factor (VEGF)and insulin like growth factor-1 (IGF-1). Using antibodies specific tothe amino terminal end (amino terminal 55 amino acids, “ATE”) orantibodies to the 18 kDa regions of the 24 kDa FGF-2, we localized theinhibition of migration to the ATE and stimulation of growth to the 18kDa domain of 24 kDa FGF-2. Thus, it was concluded that 24 kDa FGF-2affects cell behaviour differently than 18 kDa FGF-2 and that the ATEregion, which is absent from the 18 kDa FGF-2, is responsible for thisdifference.

The present invention is a truncated form of Fibroblast Growth Factor,thus it has never before been described as an independent molecule. Thefull length Fibroblast Growth Factor has both an inhibitory activity anda proliferative activity, i.e., it stimulates cells to grow which is anunwanted activity in cancer therapy. Growth factors are consideredpro-migratory and pro-angiogenic, and they can be used to stimulateangiogenesis in patients with vascular insufficiencies. The unexpectedresult of the present invention includes the separation of theinhibitory activity from the unwanted proliferative activity, thusallowing the use of a truncated growth factor as anti-angiogenic or ananti-migration compound. The present invention is not an anti-angiogenicalone, but it is effective against tumor cells. Thus, tumors that arenot susceptible to anti-angiogenic treatment will be responsive to thepresent invention.

Citation of the above documents is not intended as an admission that anyof the foregoing is pertinent prior art. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treatingdiseases and processes mediated by undesired and uncontrolled cellinvasion and/or angiogenesis by administration to a subject acomposition comprising an oligopeptide, chemical derivative orpeptidomimetic in dosage sufficient to inhibit the invasion and/orangiogenesis. The present invention is particularly useful for treatingor for suppressing the growth of tumors, the development of bloodvessels resulting in retinopathy or any other diseases dependent ofblood vessel formation. Administration of the composition to a human orsubject with prevascularized metastasized tumors will prevent the growthor expansion of these tumors.

Thus, the present invention is directed to a novel protein containingany portion of the full length 24 kDa fibroblast growth factor, theamino acid sequence of the full length 24 kDa fibroblast growth factoris shown in FIG. 8A (SEQ ID NO: 1). The present invention is directed toany portion of the full length 24 kDa fibroblast growth factor, eithercontinuous or non-continuous, or a substitution variant, additionvariant or other chemical derivative thereof that inhibits migration ofmammalian cells in vitro and reduces or completely blocks the growth ofblood vessels, tumors growth, or any physiologic or pathologic responsethat is dependent on cell migration in vivo. Preferably the ATE+31truncated protein has an amino acid sequence as shown in FIG. 8B (SEQ IDNO: 2) and the ATE+33 truncated protein has an amino acid sequence asshown in FIG. 8C (SEQ ID NO: 3). For in vivo methods, it is highlypreferable to administer a pharmaceutical composition (comprising thepolypeptide formulated in a pharmaceutically accepted diluent, adjuvant,excipient, carrier, or the like) to the subject, in an amount effectiveto modulate the migration of cells and the growth of tumor and bloodvessels in vivo.

The present invention is further directed to a pharmaceuticalcomposition useful for inhibiting the growth of tumors or angiogenesis,comprising a protein, variant or chemical derivative including apeptidomimetic or an multimeric peptide and a pharmaceuticallyacceptable carrier or excipient.

Also provided is a method for inhibiting cell migration, invasion,migration-induced cell proliferation or angiogenesis in a subject havingdisease or condition associated with undesired cell migration, invasion,migration-induced proliferation, or angiogenesis comprisingadministering to the subject an effective amount of a pharmaceuticalcomposition as described above.

In any of the foregoing methods, the disease or condition being treatedmay be primary tumor growth, tumor invasion or metastasis,atherosclerosis, post-balloon angioplasty vascular restenosis, neointimaformation following vascular trauma, vascular graft restenosis, fibrosisassociated with a chronic inflammatory condition, lung fibrosis,chemotherapy-induced fibrosis, wound healing with scarring and fibrosis,psoriasis, deep venous thrombosis, retinopathy or any another disease orcondition in which angiogenesis is pathogenic.

An effective amount of polypeptide is defined herein as that amount ofpolypeptide empirically determined to be necessary to achieve areproducible change in cell growth rate or migration, angiogenesis, ortumor size, (as determined by microscopic or macroscopic visualizationand estimation of cell doubling time, or nucleic acid synthesis assays),as would be understood by one of ordinary skill in the art. An effectivedose may be between about 1 ng/kg body weight and about 10 g/kg bodyweight, preferably between about 1 microg/kg body weight and about 100mg/kg body weight, more preferably between about 100 microg/kg bodyweight and 10 mg/kg body weight.

For methods which involve the in vivo administration of polypeptides ofthe invention, it is contemplated that the polypeptides will beadministered in any suitable manner using an appropriatepharmaceutically-acceptable vehicle, e.g., a pharmaceutically-acceptablediluent, adjuvant, excipient or carrier. Thus, the invention furtherincludes compositions, e.g., pharmaceutical compositions, comprising oneor more polypeptides of the invention. By pharmaceutical composition itis meant a composition that may be administered to a mammalian host,e.g., orally, topically, parenterally (including, but not limited tosubcutaneous injections, intravenous, intramuscular, intracisternalinjection or infusion techniques), by inhalation spray, or rectally, inunit dosage formulations containing conventional non-toxic carriers,diluents (including, but not limited to calcium carbonate, sodiumcarbonate, lactose, calcium phosphate, sodium phosphate, kaolin, water),adjuvants, vehicles, and the like, including but not limited toflavoring agents, preserving agents; granulating and disintegratingagents; binding agents; time delay materials; oils; suspending agents;dispersing or wetting agents; anti-oxidants; and emulsifiers.

The definition of polypeptides of the invention is intended to includewithin its scope variants thereof. The polypeptide variants contemplatedinclude purified and isolated polypeptides having amino acid sequencesthat differ from the exact amino acid sequences of such polypeptides byconservative substitutions, as recognized by those of skill in the art,that are compatible with the retention of the inhibitory activity ofpolypeptide. The term “variants,” when used to refer to polypeptides,also is intended to include polypeptides having amino acid additions,including but not limited to additions of a methionine and/or leadersequence to promote translation and/or secretion; additions of peptidesequences to facilitate purification (e.g., polyhistidine sequencesand/or epitopes for antibody purification); and additions ofpolypeptide-encoding sequences to produce fusion proteins. The term“variants” also is intended to include polypeptides having amino aciddeletions at the amino terminus, the carboxy terminus, or internal ofamino acids that are non-conserved amongst mammalian sequences, and thatare compatible with the retention of the inhibitory activity of thepolypeptide to which the deletions have been made.

The term “variant” also is intended to include polypeptides havingmodifications to one or more amino acid residues that are compatiblewith retaining inhibitory activity of the polypeptide. Suchmodifications include glycosylations and the addition of othersubstituents (e.g., labels, compounds to increase serum half-life (e.g.,polyethylene glycol), and the like.

In yet another aspect, the invention includes analogs of thepolypeptides of the invention. The term “analog” refers to polypeptideshaving alterations involving one or more amino acid insertions, internalamino acid deletions, and/or non-conservative amino acid substitutions(replacements). The definition of analog is intended to include withinits scope variants of analog polypeptides embodying such alterations.The term “mutant,” when used with respect to polypeptides herein, isintended to refer generically to 24 kDa FGF-2, analogs, and variants ofanalogs.

The present invention also provides purified and isolatedpolynucleotides (i.e., nucleic acids) encoding all of the polypeptidesof the invention, including but not limited to cDNAs and genomic DNAsencoding 24 kDa FGF-2 biologically active fragments thereof and DNAsencoding the same. Distinct polynucleotides encoding any polypeptide ofthe invention by virtue of the degeneracy of the genetic code are withinthe scope of the invention. The DNA sequence of the full length 24 kDafibroblast growth factor is shown if FIG. 9A (SEQ ID NO: 4), and the DNAsequence corresponding to amino acid sequence of the ATE+31 truncatedprotein of the present invention is shown in FIG. 9B (SEQ ID NO: 5), andthe amino acid sequence of the ATE+33 truncated protein is shown in FIG.9C (SEQ ID NO: 6).

Additional aspects of the invention include vectors which comprisenucleic acids of the invention; and host cells transformed ortransfected with nucleic acids or vectors of the invention. Preferredvectors of the invention are expression vectors wherein nucleic acids ofthe invention are operatively connected to appropriate promoters andother control sequences that regulate transcription and/or subsequenttranslation, such that appropriate prokaryotic or eukaryotic host cellstransformed or transfected with the vectors are capable of expressingthe polypeptide encoded thereby.

In a related aspect of the invention, host cells such as prokaryotic andeukaryotic cells, especially unicellular host cells, are modified toexpress polypeptides of the invention. Host cells may be stablytransformed or transfected with isolated DNAs of the invention in amanner allowing expression of polypeptides of the invention therein.Thus, the invention further includes a method of making polypeptides—ofthe present invention. In a preferred method, a nucleic acid or vectorof the invention is expressed in a host cell, and a polypeptide of theinvention is purified from the host cell or the host cell's growthmedium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of 24 kDa FGF-2, the peptide sequenceof 24 kDa FGF-2 (SEQ ID NO: 1), and the nucleotide sequence of 24 kDaFGF-2 (SEQ ID NO: 4).

FIG. 2 shows the effect of truncated forms of 24 kDa FGF-2 on cellmigration and proliferation. A. The effects of 24 kDa FGF-2, ATE+31 (theamino terminal end plus 31 amino acids), ATE+20 (amino terminal end plus20 amino acids), and ATE (the amino terminal end only) on MCF-7 cellmigration were tested in a Boyden Chamber assay. Proteins at 6.6×10⁻¹¹ M(open bars), 3.3×10⁻¹⁰ M (filled bars), or 8×10⁻¹⁰ M (hatched bars) wereemployed and the cell migration rates in response to 10 ng/ml IGF-1 weremeasured. Results are presented as a percent of the migration rate ofMCF-7 cells in the presence of 10 ng/ml IGF-1 alone. B. Proliferation ofMCF-7 cells by ATE+31. Cells were treated with 18 kDa FGF-2, 24 kDaFGF-2, or ATE+31 at 4×10⁻¹⁰ M and the effect on the rate of thymidineincorporation determined. Both 18 kDa FGF-2 and 24 kDa FGF-2 stimulatedthymidine incorporation 8 to 10 fold while ATE+31 had no effect. Resultsare presented relative to the rate of thymidine incorporation in theabsence of any growth factor.

FIG. 3 shows the effect of the arginine to alanine substitution withinthe amino terminal region of ATE+31 (SEQ ID NO: 2) on the inhibition ofmigration. The amino terminal 55 amino acids of ATE+31 (SEQ ID NO: 2)was divided into 4 sub-regions and the arginines in each region wereconverted to alanine by site directed mutagenesis (upper panel). Eachmutated protein was tested for inhibitory activity using MCF-7 cells and10 ng/ml IGF-1 and the results presented as a percent of the migrationrate in the presence of IGF-1 alone. The numbers on the x-axis refer tothe regions of the amino terminal peptide in which the substitutionswere made. The results show that the inhibitory activity can belocalized to regions 1 and 2.

FIG. 4 shows ATE+31 does not compete with 24 kDa FGF˜2 for binding toFGFR1. 3T3 cells were incubated at 4° C. for 2 hr with 2 ng ¹²⁵I-24 kDaFGF-2 and increasing concentrations of unlabeled 24 kDa FGF-2 or ATE+31.Values shown are the means of triplicate samples±S.D. and arerepresentative of three or more experiments. The results are presentedas a percent of maximal 24 kDa FGF-2 binding in the absence ofcompetitor.

FIG. 5 shows failure of ATE+31 to stimulate ERK phosphorylation. MCF-7and endothelial cells were treated with 1 ng/ml of 24 kDa FGF-2 or 0.37ng/ml ATE+31 for 10 min, the cells were extracted, and the proteinlysates immunoblotted with antibodies to either phospho-ERK (pERK) ortotal ERK.

FIG. 6 shows the in vivo assessment of the effect of ATE+31 onangiogenesis. 6A. Female athymic Ncr nude mice were injectedsubcutaneously at three sites near the abdominal midline with matrigel(500˜L) mixed with heparin (25 Ilg), FGF-2 (20 nM) and ATE+31 asindicated. Injection sites were chosen such that each animal received apositive control plug (FGF-2 and heparin), a negative control plug(heparin plus buffer) and a plug containing the treatment to be tested(FGF-2, heparin and ATE+31). All treatments were tested in triplicate.6B. Quantitative analysis of neovascular development in matrigel plugsin vivo. Matrigel plugs were excised from the animals, dispersed inwater and incubated at 37° C. overnight. Hemoglobin levels weredetermined using Drabkin's solution (Sigma) according to themanufacturers' instructions. The results were quantitatedspectrophotometrically at 540 nm and are presented as a function of theamount of hemoglobin present in plugs not containing added growth factor(left panel). The basal level of angiogenesis occurring in the absenceof 18 kDa FGF-2 was subtracted from the hemoglobin levels in the treatedplugs and the results presented as a percentage of that occurring in thepresence of 18 kDa FGF-2 alone (100%, right panel).

FIG. 7 shows in vivo assessment of the effect of ATE+31 on tumor growth.Two million MatLyLu prostate carcinoma cells were implanted into 0.5 mlof matrigel in the presence or absence of 400 nM ATE+31 and the gelsplaced subcutaneously into 4-8 week-old female athymic Ncr nude mice atsites near the abdominal midline. After 7 days the gels were removed,photographed (panel A) and weighed (B) Each animal was injected withMatrigel containing no cells or protein, with cells alone, and withcells and protein and the relative weight of each compared within thesame animal.

FIG. 8 shows the amino acid sequence of 8A. the full length 24 kDafibroblast growth factor, 8B. the amino acid sequence of the ATE+31truncated fibroblast growth factor, and 8C. the amino acid sequence ofthe ATE+33 truncated fibroblast growth factor.

FIG. 9 shows the nucleic acid sequence of 9A. the full length 24 kDafibroblast growth factor, 9B. the nucleic acid sequence of the ATE+31truncated fibroblast growth factor, and 9C. the nucleic acid sequence ofthe ATE+33 truncated fibroblast growth factor.

FIG. 10 shows in vivo assessment of the effect of ATE+31 on metastasis.Administration of ATE+31 inhibits the formation of metastatic foci inthe lungs of mice. MDA MB231 cells (200,000) were injected into the tailvein of SCID mice and ATE+31 administered by intravenous injection twicea week for 6 weeks. Data represents the number of foci counted per mouselung.

FIG. 11 shows effect of ATE+31 on MCF-7 tumors. Spheroids containingMCF-7 cells were placed within dorsal skinfold chambers and locally(superfused) treated with 20 ng of ATE+31 every two days. Left Panels,five days: Treated spheroids have a significantly less vascular supplyand are fragmented. Right panels, fifteen days, upper: Vascular plexusis dense in untreated and minimal in treated. Lower: Histologicalevaluation of MCR-7 tumors after 15 days of ATE+31 treatment. Tissuecontaining the tumor was removed, fixed, sectioned, and stained withhematoxylin and eosin.

FIG. 12 shows the effect of ATE+31 on Lewis Lung Carcinoma Cells (LLC).FIGS. A, B, and C show comparison of the fluoroscopy labelled LLC tumors48 hours after implantation. Before the invading blood vessels haveinfiltrated the tumor, the distribution of the fluorescently labelledcells was recorded and the area quantified. A. At implantation, B.untreated control, and C. treated with ATE+31 (100 ng/ml). The graphshows relative areas of tumors (n=3).

DETAILED DESCRIPTION OF THE INVENTION

The present inventor and his colleagues previously determined that the24 kDa, 22.5 kDa, and 22 kDa FGF-2 form of basic fibroblast growthfactor inhibit the migration of cells in culture.

The present inventor has now identified truncated forms of 24 kDa FGF-2which act as inhibitors of tumor growth, angiogenesis, and invasiveness.The proteins are potent and specific inhibitors of (a) cell invasion,(b) angiogenesis at tumor sites including sites of metastasis, (c) bloodvessel formation leading to other pathologies such as but not limited toretinopathy.

In Vitro Testing of Compositions

Migration and Growth Assays: For migration assays, MCF-7 cells orendolithial cells were harvested with trypsin, counted, centrifuged, andresuspended at 1×10⁵ cells in 0.5 ml Dulbecco's modified Eagle'smedium/0.5% bovine serum albumin. Cells were added to the upper well ofa Boyden chamber containing an 8.0-μm pore size polycarbonate membraneseparating the two chambers of a 6.5-mm Transwell (Costar). The upperwells were placed into the lower chamber containing 0.75 ml ofDulbecco's modified Eagle's medium/0.5% bovine serum albumin to which 10ng/ml of IGF-2 (Sigma) or 10 ng/ml VEGF (Sigma) was added as achemoattractant. Both chambers contained 24 kDa FGF-2 or truncated formsat the appropriate concentrations. After 4-6 hrs incubation at 37° C. in5% CO2, non-migratory cells on the upper membrane surface were removedwith a cotton swab and the cells which traversed and spread on the lowersurface of the filter were fixed and stained with Diff-Quik(Dade-Behring). The filter was mounted on a glass slide, and 4phase-contrast photomicrographs/membrane were taken at a magnificationof 100×. The number of cells per field was counted from contact sheetsand the result compared with control chamber with had no 24 kDa FGF-2added.

To measure growth rates, MCF-7 cells (6×10³) were plated in growthmedium for 48 hrs, the medium changed to assay medium containing henolred-free modified Eagle's medium supplemented with 1 mM sodium pyruvateand 0.3% lactalbumin hydrolysate plus or minus growth factors, and thecultures were allowed to incubate an additional 24 hrs. Two hrs prior tothe termination of the experiment 3H-thymidine was added. The cultureswere washed with PBS and then ice cold methanol (2×), 5% trichloraceticacid was added two times for 10 min each, and the DNA extracted with 0.3N NaOH. The number of cpm incorporated was determined by liquidscintillation.

In Vivo Study of Truncated Forms of 24 FGF-2

In vivo assessment of angiogenesis using the Matrigel Plug Assay:Ice-cold matrigel (500 μL) (Collaborative Biomedical Products, Inc.,Bedford, Mass.) was mixed with heparin (50 μg/ml), FGF-2 (400 ng/ml) andATE+31 as indicated. The matrigel mixture was injected subcutaneouslyinto 4-8 week-old female athymic Ncr nude mice at sites near theabdominal midline, 3 injections per mouse. Injection sites were chosensuch that each animal received a positive control plug (FGF-2 andheparin), a negative control plug (heparin plus buffer) and a plugcontaining the treatment to be tested (FGF-2, heparin and ATE+31). Alltreatments were tested in triplicate. Animals were sacrificed bycervical dislocation 5 days post injection. The mouse skin was detachedalong the abdominal midline and the matrigel plugs recovered and scannedimmediately at high resolution Plugs were then dispersed in water andincubated at 37° C. overnight. Hemoglobin levels were determined usingDrabkin's solution (Sigma).

In vivo assessment of the effect of ATE+31 on tumor growth. Two millionMatLyLu prostate carcinoma cells were implanted into 0.5 ml of matrigelin the presence or absence of 400 nM ATE+31 and the gels placedsubcutaneously into 4-8 week-old female athymic Ncr nude mice at sitesnear the abdominal midline. After 7 days the gels were removed,photographed and weighed. Each animal was injected with Matrigelcontaining no cells or protein, with cells alone, and with cells andprotein and the relative weight of each compared within the same animal.

Therapeutic Compositions and Methods

The preferred animal subject of the present invention is a mammal. Theinvention is particularly useful in the treatment of human subjects. Bythe term “treating” it is intended the administering to subjects of apharmaceutical composition comprising any of the truncated forms of 24kDa FGF-2 that have inhibitory activity toward cell migration leading toinhibition of tumor development and angiogenesis.

The pharmaceutical compositions of the present invention wherein thetruncated form(s) of is combined with pharmaceutically acceptableexcipient or carrier, may be administered by any means that achievetheir intended purpose. Amounts and regimens for the administration canbe determined readily by those with ordinary skill in the clinical artof treating any of the particular diseases. Preferred amounts aredescribed below.

Administration may be by parenteral, subcutaneous (sc), intravenous(iv), intramuscular, intraperitoneal, transdermal, topical or inhalationroutes. Alternatively, or concurrently, administration may be by theoral route. The dosage administered will be dependent upon the age,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

Compositions within the scope of this invention include all compositionswherein the truncated 24 kDa FGF-2 protein is contained in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. Typical dosages comprise 0.1 to 100mg/kg body weight, though more preferred dosages are described forcertain particular uses, below.

As stated above, in addition to the pharmacologically active protein,the new pharmaceutical preparations may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically as is well known in theart. Suitable solutions for administration by injection or orally, maycontain from about 0.01 to 99 percent, active compound(s) together withthe excipient.

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dissolving, or lyophilizing processes.Suitable excipients may include fillers binders, disintegrating agents,auxiliaries and stabilizers, all of which are known in the art. Suitableformulations for parenteral administration include aqueous solutions ofthe proteins in water-soluble form, for example, water-soluble salts. Inaddition, suspensions of the active compounds as appropriate oilyinjection suspensions may be administered. Suitable lipophilic solventsor vehicles include fatty oils, for example, sesame oil, or syntheticfatty acid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions that may contain substances which increase theviscosity of the suspension.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration, and all three types of formulation may be usedsimultaneously to achieve systemic administration of the activeingredient.

For topical application, the proteins of the present invention may beincorporated into topically applied vehicles such as salves orointments, which have both a soothing effect on the skin as well as ameans for administering the active ingredient directly to the affectedarea.

The carrier for the active ingredient may be either in sprayable ornonsprayable form Non-sprayable forms can be semi-solid or solid formscomprising a carrier indigenous to topical application and having adynamic viscosity preferably greater than that of water. Suitableformulations include, but are not limited to, solution, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like.If desired, these may be sterilized or mixed with auxiliary agents,e.g., preservatives, stabilizers, wetting agents, buffers, or salts forinfluencing osmotic pressure and the like. Examples of preferredvehicles for non-sprayable topical preparations include ointment bases,e.g., polyethylene glycol-1000 (PEG-1000); conventional creams such asHEB cream; gels; as well as petroleum jelly and the like.

Other pharmaceutically acceptable carriers for truncated form(s) of 24kDa FGF-2 protein according to the present invention are liposomes,pharmaceutical compositions in which the active protein is containedeither dispersed or variously present in corpuscles consisting ofaqueous concentric layers adherent to lipidic layers. The active proteinis preferably present in the aqueous layer and in the lipidic layer,inside or outside, or, in any event, in the non-homogeneous systemgenerally known as a liposomic suspension.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

Example I

Truncated forms of 24 kDa FGF-2 were generated by deletion mutagenesisthrough the placement of stop codons within the 24 kDa FGF-2 cDNA. Todetermine the effect of these truncations on the growth promotingactivity of 24 kDa FGF-2, peptides were added to MCF-7 cells and therate of cell proliferation, as determined by thymidine incorporation,compared to 24 kDa and 18 kDa FGF-2 (FIG. 2). 24 kDa FGF-2 stimulatedproliferation equally as well as 18 kDa FGF-2 (8-10-fold). However, nostimulation of proliferation was observed with either of the truncatedforms of 24 kDa FGF-2 at concentrations equal to those used for 24 or 18kDa FGF-2 (FIG. 2). Increasing the concentration of ATE+31 to 1×10⁻⁹ didnot promote proliferation. Thus, the growth stimulatory effect of 24 kDaFGF-2 is dependent on the carboxy terminal portion of 24 kDa FGF-2.However, this was not the case with the inhibition of migration.Employing the Boyden chamber assays and IGF-1 as a chemoattractant, weobserved a decrease in MCF-7 cell migration to 35.5±8% of control in thepresence of 6.6×10⁻¹¹ MATE+33 and to 22.3±5% with 3.3×10⁻¹⁰ M ATE+31.The decline in motility with 3.3×10⁻¹⁰ M ATE+31 was equal to the maximaleffect observed with full length 24 kDa FGF-2 (22.1±6%), although thetruncated protein required 5 times the concentration of the largermolecule. In both cases, increasing the concentration of the proteins byanother 5-fold resulted in a reduction in inhibitory activity. At3.3×10⁻¹⁰ M, 24 kDa FGF-2 was less effective at inhibiting migrationwhile at 8×10⁻¹⁰ M ATE+31 only inhibited migration to 61±4% of control.Deletion of additional portions of 24 kDa FGF-2 reduced the inhibitoryactivity. ATE+20 could only reduce migration to 58% at 3.3×10⁻¹⁰ M.Removal of additional amino acids had no further effect. Endothelialcell migration using VEGF as a chemoattractant was similarly affectedwith 3.3×10⁻¹⁰ M ATE+31 reducing mobility to 24% (data not shown). Theseresults demonstrate that the inhibition of migration activity of 24 kDaFGF-2 is localized to the amino terminal end and does not require eitherthe published receptor binding sites nor the heparin binding sites foundwithin 18 kDa FGF-2.

Example II

Further attempts to determine if the inhibitory activity was dependenton specific regions within the ATE involved arginine to alaninesubstitution. Because of the large number of arginines in the ATE, thesequence was separated into 4 regions each containing 3 or 4 arginines(FIG. 3). Each region was modified separately and the effect oninhibition of migration was tested and compared to the unmodifiedATE+31. Conversion of arginine to alanine in the two regions at thecarboxy terminal end of the ATE (3 and 4) had little effect on themigration rates; these molecules still inhibited migration by 70 to 75%.However, arginine to alanine substitutions within either of the tworegions at the amino terminal end (1 and 2) reduced the inhibitoryactivity of ATE+31; cell migration was 50% of untreated cultures asopposed to 20% for the wild type ATE+31. The inhibition of migrationcould be reduced even further by combining regions 1 and 2 resulting inless than 15% inhibition of migration.

Example III

The localization of the inhibitory activity to the amino terminal end of24 kDa FGF-2 suggests that there is some, interaction between thisportion of the protein and the cells it is affecting. In previousstudies, it was shown that the FGF receptor to which 24 kDa FGF-2 bindsin endothelial, MCF-7, and 3T3 cells is FGFR1. To determine whetherATE+31 contains a major binding domain for interaction with FGFR1,competition binding experiments were performed with iodinated 24 kDaFGF-2 vs. unlabeled 24 kDa FGF-2, 18 kDa FGF-2, or ATE+31 (FIG. 4).Competition between labeled 24 kDa FGF-2 and itself resulted in a dosedependent decline in the binding of labeled protein with an 81±3%decrease in binding at a 100-fold excess. At this concentration, 18 kDaFGF-2 also caused a similar reduction in the binding of the 24 kDa FGF-2(79±5%). However, ATE+31 had no significant effect on the binding of¹²⁵I-24 kDa FGF-2, even at a 1000-fold excess, suggesting that no majorFGFR1 binding sites are found within the amino terminal portion of 24kDa FGF-2. To determine if ATE+31 had the ability to activate an FGFR1regulated signalling pathway, its effect on ERK1/2 activation wasanalysed using phospho-specific antibodies (FIG. 5). In both MCF-7 cellsand endothelial cells, 24 kDa FGF-2, at a concentration of 4×10⁻¹¹ M,stimulated the phosphorylation of ERK 1/2, a response which also occursin 3T3 cells. However, at the same molar concentration, ATE+31 failed toaffect ERK1/2 phosphorylation in these cells. Increasing theconcentration to 4×10⁻¹⁰ M had no effect on the level of ERKphosphorylation.

Example IV

The effect of ATE+31 on angiogenesis was tested directly by implantingmatrigel plugs infused with vehicle, 4×10⁻¹¹ M 18 kDa FGF-2, and 18 kDaFGF-2 plus 15 or 150 nM ATE+31 into mice and measuring the degree ofvascular formation. In the presence of FGF-2 alone, there was a robustangiogenic response as indicated by the pink hue distributed throughoutthe plug (FIG. 6). In the presence of 15 nM ATE+31, the amount ofvascular development was reduced in all three mice while at 150 nM theamount of vascularization was indistinguishable from the plugscontaining no 18 kDa FGF-2 (FIG. 6 a). To obtain a more quantitativeanalysis of the effects of ATE+31 on angiogenesis, the hemoglobincontent within each plug was measured. In the presence of 18 kDa FGF-2alone, the amount of hemoglobin increased 3.2-fold over the controlplugs containing no added growth factor (FIG. 6 b). The addition of 15mM ATE+31 reduced this increase to 2.6 times control values while in thepresence of 150 nM ATE+31 the increase was only 1.5 times controlvalues. Thus, the net increase in vascular development with the higherconcentration of ATE+31 was only 22% of that in plugs containing 18 kDaFGF-2 alone.

Example V

The effect of the protein on the growth of tumors was studied usingmatrigel plugs impregnated with 2 million MatLyLu rat prostate tumorcells in the presence or absence of 400 nM ATE+31. Plugs were implantedand ATE+31 added to the plug which remained in the animal for 7 days,removed, and then weighed. In FIG. 7A, the matrigel plugs removed fromthree different mice show a significant reduction in vascularization inthe ATE+31-treated animals (as indicated by the red color). The averageweight of the plugs is presented in FIG. 7B. The weight of untreatedMatLyLu-matrigel was 0.90±0.22 grams versus 0.56±0.19 grams for theATE+31 treated plugs. If the average starting weight of the Matrigelplugs (no cells or ATE+31) is subtracted from these values, the netreduction in tumor size in the presence of ATE+31 is 40±18%.

Example VI

The skinfold chamber model was used to test the effect of ATE+31 on theangiogenic response to tumor development in vivo (FIG. 11). Thistechnique allows for the continuous measurement of the changes in bothrumor size and vascular density within a single animal over a prolongedperiod of time. Tumor spheroids with similar diameters (600-1,000 μm)formed with MCF-7 cells implanted into skinfold chambers and twenty-fourhours and every two days after, 20 μl of a 1 μg/ml solution of ATE+31(20 ng) was added directly to the spheroid. At day 5, there was alreadya significant difference in the density of the neovasculature within thearea of the spheroid, the untreated are showed an extensive network ofblood vessels in contrast to the limited response in the treatedanimals. In addition to the difference in the vascular density, theintegrity of the growing vessels was also affected by the presence ofthe peptide. Higher magnification shows that the treated area containsfragment vessels (arrows). Video analysis of blood flow through thesevessels revealed a diminished rate of flow, an effect we attribute tothe formation of poorly differentiated blood vessels. These differenceswere further magnified by day 15. By day 15, the vascular plexus in theuntreated animals is extremely dense, filling up most of the spacewithin the spheroid while the treated animals have very few vessels inthat area at all. Histological evaluation of the skin at day 15 showsthe extent of the suppression of tumor growth by ATE+31. Comparison ofthe tumors indicated by dark blue area shows a dramatic difference insize. The average size of the treated tumor spheroids was only 7.8% ofthe untreated (1.3 mm² vs. 16.8 mm²; n=3) after 15 days.

Example VII

In addition to the experiments with the mammary tumor cells, we havetested the peptide against Lewis Lung Cancinoma (LLC) cells. The purposeof these experiments was to determine whether ATE+31 would be effectiveagainst a fast growing, more aggressive tumor cell (FIG. 12). Over thefirst 7 days, untreated tumor spheroid (♦) grew by 9-fold while thetreated spheroids (▪) increased by 3 times. The next three days saw asignificant increase in the rate of tumor expansion in untreated animalswith a final volume 28 times larger than that of the original spheroid.In contrast, ATE+31 suppressed rumor growth and the treated tumor volumewas only 19% of the control. This was reflected in the vascularizationof the tumor. At 6 days after implantation, a dense network was seen inuntreated animals while the area containing angiogenic blood vessels inATE+31 treated animals was small, diffuse, and poorly developed.

Determining whether ATE+31 can suppress the growth of tumorsindependently of its inhibition of angiogenesis was made possible by therapid rate of LLC cell growth. This allows for a measurable increase inspheroid size prior to the infiltration of angiogenic blood vessels intothe tumor itself (which occurs about 2-3 days after implantation. FIG.12 shows that spheroids containing fluorescent-labeled cells appear asan intensely fluorescent body with distinct edges and an average area of6.9±0.5 mm² (FIG. 12A). After 48 hour, the spheroid in control animalshas spread making a larger and very diffuse structure with no inner coreof the spheroid still visible (FIG. 12B; area=19.8 mm²). However,treatment with ATE+31 mitigates the expansion and the spheroid bodyremains intact (FIG. 12C). No significant changes in are was observed(6.1±1.6 mm²). Thus, suppression of tumor growth can occur even in theabsence of a vascular system indicating that ATE+31 is more than ananti-angiogenic molecule.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

REFERENCE LIST

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1. An isolated nucleic acid molecule comprising the nucleic acidmolecule of SEQ ID NO: 5, wherein the nucleic acid molecule encodes fora polypeptide having at least one of anti-angiogenic, tumor suppressiveor anti-migratory activity, but does not have stimulation of cellproliferation activity.
 2. An isolated nucleic acid molecule 95%identical to SEQ ID NO: 5, wherein the nucleic acid molecule encodes aprotein having at least one of anti-angiogenic, tumor suppressive oranti-migratory activity, but does not have stimulation of cellproliferation activity.
 3. The isolated nucleic acid molecule of claim1, wherein said nucleic acid molecule is covalently linked to adetectable group.
 4. The isolated nucleic acid molecule of claim 3,wherein said detectable group is selected from the group consisting ofradiolabel and fluorescent group.
 5. The isolated nucleic acid moleculeof claim 1, wherein said nucleic acid molecule is operably linked to oneor more expression control elements.
 6. A vector comprising the nucleicacid of claim
 1. 7. A recombinant host cell, comprising the vector ofclaim 6, whereby said cell is capable of expressing said nucleic acid.8. The host cell of claim 7, wherein said host is selected from thegroup consisting of prokaryotic and eukaryotic hosts.
 9. A process forthe production of a polypeptide having at least one of anti-angiogenic,tumor suppressive or anti-migratory activity, but does not havestimulation of cell proliferation activity, said process comprising: a)growing under suitable nutrient conditions host cell transformed ortransfected with the nucleic acid molecule of SEQ ID NO: 5; and b)isolating the polypeptide products of the expression of the nucleic acidmolecule.